Lyophilized hgf preparations

ABSTRACT

The invention relates to a lyophilized HGF preparation prepared by lyophilizing an aqueous solution containing HGF, and a lyophilized HGF preparation containing a stabilizer, sodium chloride, a buffer, and/or a surface active agent. According to the invention, HGF can be stabilized, and it can be stored for a long period.

TECHNICAL FIELD

[0001] The present invention relates to a lyophilized HGF preparationobtained by lyophilizing a solution containing HGF (hepatocyte growthfactor). More particular, it relates to the lyophilized HGF preparationcontaining at least one of stabilizer, sodium chloride, buffer orsurface active agent. The invention hence presents a stabilizedpreparation of HGF that can be stored for a long period.

BACKGROUND ART

[0002] HGF is a protein that enhances proliferation of liver parenchymacells, and proteins having different amino acid sequences have beenreported, and are known in the names of HGF, TCF, SCF, etc. In theinvention, these known proteins having hepatocyte growth activity arecollectively called HGF. HGF is a physiological active peptide showingvarious pharmacological actions, and its pharmacological actions arereported, for example, in Experimental Medicine (Japan), Vol. 10, No. 3(extra issue), 330-339 (1992). Owing to its pharmacological actions, HGFis expected to be developed as agent for liver cirrhosis, agent forkidney disease, epithelial cell growth promoter, carcinostatic agent,side effect inhibitor for cancer therapy, agent for lung disorder, agentfor gastroduodenal lesion, agent for cerebral and nervous disorder,agent for relieving side effects caused by immunosuppressants, collagendecomposition promoter, agent for cartilage disorder, agent for arterialdisease, agent for lung fibroid, agent for liver disease, agent forabnormal blood clotting, agent for hypoproteinemia, wound cure agent,improving agent for nervous disorder, hematopoietic stem cell promoter,hair growth promoter, etc. (Japanese Laid-open Patent No. 4-18028,Japanese Laid-open Patent No. 4-49246, EP No. 492614, Japanese Laid-openPatent No. 6-25010, WO 93/8821, Japanese Laid-open Patent No. 6-172207,Japanese Laid-open Patent No. 7-89869, Japanese Laid-open Patent No.6-40934, WO 94/2165, Japanese Laid-open Patent No. 6-40935, JapaneseLaid-open Patent No. 6-56692, Japanese Laid-open Patent No. 7-41429, WO93/3061, Japanese Laid-open Patent No. 5-213721, etc. ).

[0003] Preparations of HGF are disclosed in WO 90/10651 and JapaneseLaid-open Patent No. 6-247872. This publication of WO 90/10651 disclosesa deletion type HGF (dLeHGF) deleting five residues of amino acid fromHGF, and it is named TCFII. This specification shows that HGF isstabilized by albumin, human serum, gelatin, sorbitol, mannitol,xylitol, etc. But, it relates to aqueous solution preparations, and HGFis stabilized in an aqueous solution. The publication of JapaneseLaid-open Patent No. 6-247872 unveils a preparation having HGF containedat high concentration by coexistence of basic amino acids and HGF (TCF).

[0004] Generally, the protein is not so stable in freezing operation(Protein, Nucleic Acid, Enzyme (Japan), 37(9), 1517, 1992). Thestabilizer of protein in an aqueous solution is intended to stabilize bymutual action of water molecule and protein. Therefore, in a lyophilizedpreparation of protein in the absence of water, the stabilizer ofprotein for an aqueous solution shows no stabilizing effect in mostcases (Protein, Nucleic Acid, Enzyme (Japan), 37(9), 1517, 1992).

[0005] On the other hand, nothing has been known about lyophilized HGFpreparation, and it could not expected how far the lyophilized HGFpreparation would show physical and biological stability.

[0006] The aqueous solution preparation of HGF itself is, when stored atlow temperature or room temperature for several days, changed inproperties, showing aggregation, turbidity and gelation, and formsvariants and polymers, and it is low in physical stability and islowered in biological activity, and hence it is low in stability ofbiological activity and is not a stable preparation suited long-termstorage. It has been a fatal point for development of HGF as medicinesor animal drugs in a form of injection preparation. The invention solvesthe above-mentioned problems. That is, it is an object of the inventionto present a stable preparation which can store for a long period asmedicines for medical treatment or animal drugs.

DISCLOSURE OF THE INVENTION

[0007] The invention relates to a lyophilized HGF preparation. Thislyophilized HGF preparation may contain a stabilizer such as glycine,alanine, sorbitol, mannitol, and dextran sulfate, or may contain abuffer such as citrate.

[0008] Other invention of the present invention relates to a lyophilizedHGF preparation containing stabilizer, sodium chloride, buffer andsurface active agent.

[0009] In the lyophilized HGF preparation of the invention, HGF isstabilized and can be stored for a long period.

THE BEST MODE FOR CARRYING OUT THE INVENTION

[0010] As HGF used in the present invention, there can be used one whichprepared by various methods if it is purified to an extent that it canbe used as a medicine.

[0011] Various methods are known for preparing HGF. For example, HGF canbe obtained by extraction and purification from organs (e.g. liver,spleen, lung, bone marrow, brain, kidney, placenta, etc.), blood cells(e.g. platelet, leucocyte, etc.), serum and plasma of mammals such asrat, cow, horse, sheep and the like (see FEBS Letters, 224, 312, 1987;Proc. Natl. Acad. Sci. USA, 86, 5844, 1989, etc.).

[0012] Also, it is possible to obtain HGF by cultivation of primaryculture cells or cell lines producing HGF, followed by separation andpurification from the culture product (e.g. culture supernatant,cultured cell, etc.). Further, HGF can be obtained by gene engineeringmethod which comprises cloning the gene coding HGF with a proper vector,inserting it into a proper host cell to give a transformant, andseparating the desired recombinant HGF from the culture supernatant ofthe transformant (e.g. Nature, 342, 440, 1989, Japanese Laid-open PatentNo. 5-111383, Biochem. Biophys. Res. Commun., 163, 967, 1989). The hostcell is not specifically limited, and various host cells conventionallyused in gene engineering methods can be used, which are, for example,Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, and plantor animal cells.

[0013] More specifically, the method of extracting and purifying HGFfrom live tissues is, for example, to administer carbon tetrachloride toa rat intraperitoneally, remove a liver from the rat with hepatitis,grind it, and purify by the ordinary protein purifying technique such asgel column chromatography using S-Sepharose and heparin Sepharose, HPLCand the like.

[0014] Further, by the gene engineering method, the gene coding theamino acid sequence of human HGF is cloned into a vector such as bovinepapilloma virus DNA and the like to obtain an expression vector, and byusing this expression vector, animals cells such as Chinese hamsterovary (CHO) cells, mouse C127 cells, monkey COS cells and the like aretransformed, and HGF can be obtained from the culture supernatant of thetransformants.

[0015] In thus obtained HGF, a part of the amino acid sequence of HGFmay be deleted or substituted by other amino acid(s), another amino acidsequence may be inserted, one or more amino acids may be bonded to theN-terminal and/or C-terminal, or saccharide chain(s) may likewise bedeleted or substituted, providing it has substantially the same effectas HGF.

[0016] The “lyophilized HGF preparation” refers to a preparationprepared by lyophilizing an aqueous solution containing HGF by use of anordinary lyophilizing method.

[0017] The “stabilizer” includes amino acids (e.g. glycine, alanine,etc.), polysaccharides (e.g. heparin, dextran sulfate, etc.), sugaralcohols (e.g. sorbitol, mannitol, etc.) and the like, and two or moretypes thereof may be used simultaneously. The lyophilized HGFpreparation prepared by adding the stabilizer is a preparation furtherincreased in storage stability of HGF. Preferred stabilizers areglycine, alanine, sorbitol, mannitol, and dextran sulfate. For example,a preferred adding amount of glycine, alanine, sorbitol or mannitol is0.01 to 100 times by weight of the weight of HGF, and more preferably0.1 to 10 times by weight.

[0018] The “buffer” includes, for example, phosphate buffer and citratebuffer. The buffer acts to adjust the pH of the aqueous solution afterre-dissolving, and keep the solubility of HGF. That is, for example, inthe case of the recombinant HGF used in Examples, the solubility of HGFvaries with the pH, and the solubility is about 0.1 to 5.0 mg/ml aroundpH 7, but the solubility is over 20 mg/ml around pH 5, and therefore itis preferred to keep the pH around 5.0 to 6.0. A preferred buffer is acitrate buffer, and more preferably sodium citrate buffer is used. Thiscitrate buffer also contributes to stabilization of HGF in an aqueoussolution after re-dissolving. A preferred range of adding the buffer is,for example, 1 to 100 mM to the amount of water after re-dissolving.

[0019] The “surface active agent” includes, for example, polysorbate 20,polysorbate 80, pluronic F-68, and polyethylene glycol, and two or moretypes thereof may be used simultaneously. A particularly preferredsurface active agent is polysorbate 80. It is known that HGF is likelyto be adsorbed on a container material such as glass and resin.Therefore, by adding a surface active agent, adsorption of HGF to afterre-dissolving to the container is prevented. A preferred range of addingamount of surface active agent is 0.001 to 2.0% by weight, for example,to the weight of water after re-dissolving.

[0020] The “sodium chloride” acts to keep solubility of HGF. That is,for example, in the case of recombinant HGF used in Examples, thesolubility is enhanced by adding sodium chloride, and the solubility isnotably increased in particular at 300 mM or more (Japanese Laid-openPatent No. 6-247872). An amount of addition of sodium chloride islimited by the osmotic pressure ratio, but it may be an amount showingan osmotic pressure ratio of injection preparation for general use. Inparticular, the osmotic pressure ratio is preferred to be 1 to 2 whichis permitted as the osmotic pressure ratio of injection for medicaltreatment or animal drug, and it is preferred to add, for example, by150 to 300 mM to the amount of water after re-dissolving.

[0021] The lyophilized HGF preparation is prepared by lyophilizing anaqueous solution containing HGF by an ordinary lyophilizing method. Forexample, HGF is dissolved in a proper solvent (for example, sterilizedwater, buffer, physiological saline, etc.), filtered through a filter tobe sterilized, and, if necessary, stabilizer, buffer, surface activeagent, sodium chloride and others may be added, and the mixture islyophilized. The preparation of the invention may contain additivesnecessary for pharmaceutical manufacturing, for example, a dissolvingaid, an antioxidant, a pain-alleviating agent, an isotonic agent, andthe like. The lyophilizing method may comprise three unit operations,for example, (1) a freezing step of cooling and freezing under ordinarypressure, (2) a first drying step of sublimating and drying free waternot restrained by solute under reduced pressure, and (3) a second dryingstep of removing the intrinsic adsorbed water and crystal water ofsolute (Pharm. Tech. Japan, 8(1), 75-87, 1992). HGF is very stable whenpreparing a solution, when lyophilizing, and in an aqueous solution byre-dissolving the lyophilized preparation. The content of HGF may beproperly adjusted depending on the disease to be treated and route ofadministration.

[0022] The lyophilized preparation is used by adding distilled water forinjection and re-dissolving, before use.

INDUSTRIAL APPLICABILITY

[0023] The lyophilized HGF preparation of the invention can stabilizeHGF, and can be stored for a long period.

EXAMPLES

[0024] The invention is further described by presenting Examples, but itmust be noted that the invention is not limited to these Examples alone.In the Examples, dLeHGF (five-amino acid depletion type HGF, also knownas TCFII) disclosed in the publication of WO 90/10651 was used.

Example 1 Preparation of Lyophilized HGF Preparation

[0025] In 10 mM citrate buffer (pH 5.0) containing 300 mM sodiumchloride and 0.01% polysorbate 80, HGF was dissolved by 20 mg/ml, and anaqueous solution of HGF was obtained aseptically. After adjusting the pHof the aqueous solution, it was aseptically charged into a vial, andlyophilized in the condition as shown in Table 1, and a lyophilized HGFpreparation was obtained. The arrow mark (→) in the table shows thetemperature is changed. TABLE 1 Freezing First drying Second drying stepstep step Temperature (° C.) 5 → −40 −40 −40 → 0  0 0 → 20 20 Time (hr) 1  10  8 24  1 24 Pressure (mmHg) 760 760 <1 <1 <1 <1

Example 2 Preparation of Lyophilized HGF Preparation

[0026] A lyophilized HGF preparation was obtained by using 10 mM citratebuffer (pH 6.0) instead of 10 mM citrate buffer (pH 5.0) in Example 1.

Example 3 Preparation of Lyophilized HGF Preparation

[0027] A lyophilized HGF preparation was obtained by using 10 mMphosphate buffer (pH 6.0) instead of 10 mM citrate buffer (pH 5.0) inExample 1.

Example 4 Preparation of Lyophilized HGF Preparation

[0028] A lyophilized HGF preparation was obtained by using 10 mMphosphate buffer (pH 7.0) instead of 10 mM citrate buffer (pH 5.0) inExample 1.

Example 5 Preparation of Lyophilized HGF Preparation

[0029] In 10 mM citrate buffer (pH 5) containing 300 mM sodium chlorideand 0.01% polysorbate 80, HGF was dissolved by 20 mg/ml. In succession,glycine was dissolved by 50 mg/ml, and a dissolved solution of HGF wasobtained aseptically. After adjusting the pH of the dissolved solution,it was aseptically charged into a vial, and lyophilized in the samecondition as in Example 1 and a lyophilized HGF preparation wasobtained.

Example 6 Preparation of Lyophilized HGF Preparation

[0030] A lyophilized HGF preparation was obtained by using alanineinstead of glycine in Example 5.

Example 7 Preparation of Lyophilized HGF Preparation

[0031] In 10 mM citrate buffer (pH 5) containing 300 mM sodium chlorideand 0.01% polysorbate 80, HGF was dissolved by 20 mg/ml. In succession,sorbitol was dissolved by 200 mg/ml, and a dissolved solution of HGF wasobtained aseptically. After adjusting the pH of the dissolved solution,it was aseptically charged into a vial, and lyophilized in the samecondition as in Example 1 and a lyophilized HGF preparation wasobtained.

Example 8 Preparation of Lyophilized HGF Preparation

[0032] In 10 mM citrate buffer (pH 6) containing 300 mM sodium chlorideand 0.01% polysorbate 80, HGF was dissolved by 10 mg/ml. In succession,dextran sulfate was dissolved by 50 mg/ml, the pH was adjusted, and adissolved solution of HGF was obtained. It was then charged into a vial,and lyophilized in the same condition as in Example 1 and a lyophilizedHGF preparation was obtained.

Example 9 Preparation of Lyophilized HGF Preparation

[0033] A lyophilized HGF preparation was obtained in the same manner asin Example 1, except by using 10 mM citrate buffer (pH 6.0) instead of10 mM citrate buffer (pH 5.0), and regulating HGF concentration at 10mg/ml.

Test Example 1 Effects of Lyophilizing Process on Biological Activity ofHGF

[0034] To observe changes in biological activity of HGF in thelyophilizing process, using HGF aqueous solution before lyophilizationand HGF aqueous solution re-dissolved directly after lyophilization inExample 1, the biological activity of HGF was measured (the measuringmethod of biological activity is shown below). The results are shown inTable 2. Since the specific activity was not changed before and afterlyophilization, it is shown that the biological activity of HGF is notinactivated by the lyophilizing process and re-dissolving, whichsuggests that HGF is usable as a lyophilized preparation.

Measuring Method of Biological Activity

[0035] Hepatocytes obtained by liver perfusion of male Wistar rats werepurified, and, after confirming the cell survival rate, seeded on aplate at 1×10⁴/well. After pre-incubation for 20 hours in 5% carbondioxide incubator, HGF sample and standard sample were added (n=3).After further pre-incubation for 24 hours in 5% carbon dioxideincubator, [³H-thymidine] was added to label for 2 hours. Cells werecollected by a cell harvester, and the amount of [³H] taken into cellswas measured. Results of measurement were verified by a parallel linecalibration method, and the specific activity to the standard sample wasdetermined. TABLE 2 Biological activity before and after lyophilizationSample Specific activity Solution preparation before 0.89 lyophilizationLyophilized preparation immediately 0.94 after re-dissolving

Test Example 2 Properties after Dissolving Lyophilized Preparation

[0036] Lyophilized preparations prepared in Examples were stored for 1month at −40° C., 25° C., and 50° C., and dissolved, and properties ofthe dissolved preparations were observed visually. The lyophilizedpreparation was dissolved by using purified water. Results are shown inTable 3. When stored at −40° C. or 25° C., the preparations of allExamples were stable in the properties. When stored at 50° C., thepreparation in Example 1 was turbid immediately after dissolving, butpreparations of Examples 5, 6 and 7 were stable in properties. TABLE 3Properties after dissolving lyophilized preparations (stored for 1month) Properties Preparation −40° C. 25° C. 50° C. Example 1 ClearClear Turbid Example 5 Clear Clear Clear Example 6 Clear Clear ClearExample 7 Clear Clear Clear

Test Example 3 Polymer Content Changes in Lyophilized Preparations

[0037] Lyophilized preparations prepared in Examples 1, 5, 6 and 7 werestored for 1 month or 2 months at −40° C., 25° C., 40° C., and 50° C.,and the ratio of polymer content and HGF content contained in thelyophilized preparations were measured. The measuring method is the gelfiltration method as explained below. Results are shown in Table 4 andTable 5. Regardless of the storage temperature, a polymer production waslow in the preparations of all Examples, and the preparations werestable physically. In particular, the polymer production was extremelysmall in the preparations of Examples 5, 6 and 7, and the preparationswere stable physically.

Measuring Method of Polymer Content

[0038] The concentration of HGF was diluted to 2 mg/ml, and was measuredin the following conditions by the gel filtration method.

[0039] Column: TOSOH TSK G-3000SW XL (φ0.78×30 cm)

[0040] Flow velocity: 0.5 ml/min

[0041] Detection: OD 280

[0042] Temperature: 25° C.

[0043] Carrier: 10 mM Tris, 150 mM NaCl, 0.05% SDS, pH 7.0

[0044] Application: 20 μl

[0045] Retention time of polymer: 13.0 min

[0046] Retention time of HGF: 14.4 min TABLE 4 Polymer content/HGFcontent in lyophilized preparations stored for 1 month −40° C. 25° C.40° C. 50° C. Example 1 1.07% 1.59% 2.76% 6.17% Example 5 0.92% 1.39%1.83% 4.09% Example 6 0.93% 1.54% 1.81% 2.90% Example 7 0.90% 1.35%2.57% 6.64%

[0047] TABLE 5 Polymer content/HGF content in lyophilized preparationsstored for 2 months −40° C. 25° C. 40° C. 50° C. Example 1 0.92% 1.44%3.91% 12.23%  Example 5 0.88% 1.21% 2.49% 7.49% Example 6 0.85% 1.10%1.96% 5.76%

Test Example 4 Effects of Dextran Sulfate on Polymer Production

[0048] The lyophilized preparation prepared in Example 8 was stored for1 month at 50° C., and the ratio of polymer content and HGF contentcontained in the lyophilized preparations were measured. The measuringmethod was same as in Test example 3. As a comparative example, thelyophilized preparation of Example 9 prepared in the same compositionand method except that dextran sulfate was not contained was used andtested similarly. The results are shown in Table 6. As shown in Table 6,by adding dextran sulfate, it has been found that the polymer productionwas low even if stored at high temperature, and that the stability isenhanced. TABLE 6 Polymer content/HGF content of lyophilizedpreparations Before start of After storage for storage 1 month at 50° C.Example 8 2.46%  9.45% Example 9 1.78% 14.01%

Test Example 5 Changes of Biological Activity of LyophilizedPreparations

[0049] Lyophilized p reparations prepared in Examples 1, 5, 6 and 7 werestored for 1 month or 2 months at −40° C., 40° C., 50° C. and 60° C.,and the biological activity of the aqueous solution after re-dissolvingthe lyophilized preparations was measured by the biological activitymeasuring method shown in Test example 1. The results are shown in Table7 and Table 8. The initial values of biological activity of aqueoussolutions after re-dissolving the preparations in Examples 1, 5, 6 and 7were respectively 1.01±0.25, 0.91±0.18, 0.88±0.05, and 1.03±0.04. Whenstored at 60° C., a slightly lowering tendency was noted in thebiological activity, but when stored at 50° C. or lower temperature,there was almost no change in the biological activity in thepreparations of any Example, and the biological activity was stable.TABLE 7 Biological activity of lyophilized preparations stored for 1month (specific activity) −40° C. 40° C. 50° C. 60° C. Example 1 0.96 ±0.13 0.92 ± 0.13 0.81 ± 0.07 0.54 ± 0.05 Example 5 0.80 ± 0.14 0.99 ±0.10 0.80 ± 0.16 0.72 ± 0.03 Example 6 0.92 ± 0.14 1.02 ± 0.06 0.94 ±0.08 0.78 ± 0.03 Example 7 0.92 ± 0.02 0.97 ± 0.04 0.83 ± 0.06 —

[0050] TABLE 8 Biological activity of lyophilized preparations storedfor 2 months (specific activity) −40° C. 40° C. 60° C. Example 1 1.14 ±0.14 0.98 ± 0.01 0.46 ± 0.09 Example 5 0.95 ± 0.05 0.84 ± 0.09 0.57 ±0.01 Example 6 1.11 ± 0.14 1.09 ± 0.03 0.52 ± 0.02

1. A lyophilized HGF preparation.
 2. The lyophilized HGF preparation ofclaim 1 , wherein the preparation contains a stabilizer.
 3. Thelyophilized HGF preparation of claim 2 , wherein the stabilizer isglycine, alanine, sorbitol, mannitol, or dextran sulfate.
 4. Thelyophilized HGF preparation of any one of claims 1 to 3 , wherein thepreparation contains a buffer.
 5. The lyophilized HGF preparation ofclaim 4 , wherein the buffer is a citrate buffer.
 6. A lyophilized HGFpreparation which contains a stabilizer, sodium chloride, a buffer, anda surface active agent.